| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-cells: activation of ATP-sensitive K+ channels
MS 1201 Received 6 June 2000; accepted after revision 1 September 2000.
 |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
-cells through a negative feedback loop, involving both KATP and voltage-gated conductances.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Zinc, an essential element present in almost all tissues, is a co-factor or component of at least 300 identified metalloenzymes, as well as a structural component of several non-enzymatic proteins (for review see Vallee & Falchuk, 1993). In the central nervous system, Zn2+ has been localised in nerve terminals and synaptic vesicles of excitatory neurons (Perez-Clausell & Danscher, 1985; Slomianka, 1992). The transition ion is taken up and released upon nerve excitation along with neurotransmitters (Assaf & Chung, 1984; Howell et al. 1984), and has been proposed to act as a modulator of synaptic transmission and neuronal death (Nishimura, 1988; Wang & Quastel, 1990; Xie & Smart, 1991; Vautrin et al. 1993; Dunant et al. 1996; Choi & Koh, 1998). Zn2+ also alters the behaviour of various ligand-gated and voltage-gated ion channels (for review see Harrison & Gibbons, 1994; Smart et al. 1994).
In pancreatic -cells, Zn2+ is involved in different steps of insulin biosynthesis, processing and storage. In all but a few species, insulin is stored in membrane-limited granules in the form of Zn2+-insulin crystals (see Blundell et al. 1971; Dodson & Steiner, 1998). As conversion of pro-insulin to insulin proceeds, Zn2+ favours the formation of hexamers of insulin and promotes its crystallization. The concentration of Zn2+ in the granules has been estimated at about 20 mM (Hutton et al. 1983; Foster et al. 1993). Since Zn2+-insulin hexamers dissociate rapidly (within seconds) after exposure of the granule interior to the extracellular milieu (Gold & Grodsky, 1984), high concentrations of unbound Zn2+ are likely to be produced locally.
It is conceivable, therefore, that released Zn2+ alters the function of pancreatic -cells by acting on ion channels. Inhibitory effects of micromolar concentrations of Zn2+ on insulin secretion or electrical activity of -cells have indeed been reported (Ghafghazi et al. 1981; Ferrer et al. 1984; Aspinwall et al. 1997), but they were interpreted only in terms of a block of voltage-dependent Ca2+ channels, interaction with intracellular Ca2+, or common-ion effect on Zn2+-insulin dissociation. However, other targets can be envisaged for this effect, such as the ionic permeabilities linking stimulation by glucose to insulin secretion. In the present study, we analysed the effects of Zn2+ on ionic conductances in RINm5F cells (clonal insulinoma cells). Besides a blocking effect on voltage-activated Ca2+ currents, we found, surprisingly, that micromolar Zn2+ concentrations enhanced the open-state probability of ATP-sensitive K+ (KATP) channels, therefore regulating the resting membrane potential and excitability of the cells.
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cell culture
Cells from the insulin-producing cell line RINm5F, derived from a rat islet cell tumour (Praz et al. 1983), were cultured in a humidified 5 % CO2-95 % air atmosphere at 37°C, in RPMI 1640 medium (Gibco BRL, Life Technologies AG, Basel, Switzerland) supplemented with 10 % fetal calf serum, 11 mM glucose, 100 U ml-1 penicillin and 100 µg ml-1 streptomycin. Three to seven days before experiments, cells were seeded out onto Falcon 3001-type Petri dishes (Becton Dickinson UK Ltd, Plymouth, UK) at a density of 100000-200000 cells per dish.
Electrophysiology and solutions
Both whole-cell and outside-out configurations of the patch-clamp technique were used. Whole-cell recordings were performed using fired-polished electrodes, pulled from borosilicate glass, and showing an open resistance of 2-3 M
. Signals were amplified using an Axopatch 200B amplifier and filtered through a four-pole low-pass Bessel filter at 1 or 2 kHz, before digitisation with a Digidata 1200 interface and analysis with pCLAMP 8 software (Axon Instruments, Inc., Foster City, CA, USA). Capacitative transients and series resistance were compensated (> 70 %), using the circuitry incorporated to the amplifier. The following solutions were used. For calcium currents, bath solution (mM): 130 NaCl; 3 KCl; 10 CaCl2; 2 MgCl2; 10 Hepes; 10 D-glucose, pH 7·2 (NaOH) containing 200 nM tetrodotoxin (TTX); pipette solution (mM): 145 CsCl; 1 MgCl2; 10 Hepes; 1 CaCl2; 12 EGTA; 1 Mg-ATP, pH 7·2 (NaOH). For potassium currents and current-clamp experiments, bath solution (mM): 145 NaCl; 3 KCl; 2 CaCl2; 2 MgCl2; 10 Hepes; 10 D-glucose, pH: 7·2 (NaOH); pipette solution (mM): 10 NaCl; 140 KCl; 1 MgCl2; 10 Hepes; 1 EGTA; 1 Mg-ATP; pH 7·2 (KOH). The concentration of Mg2+ in the patch pipette was adjusted to 2 mM with MgCl2 when the Mg-ATP concentration was reduced. Compounds were directly dissolved into the bath solutions, except for those containing tetraethylamonium (TEA) plus 4-aminopyridine (4-AP), which were obtained by equimolar substitution with NaCl. For voltage-dependent currents, the P/4 procedure was used to subtract leakage currents, except in the case of Ca2+ currents elicited by voltage ramps, which were determined by digital subtraction of cadmium (250 µM)-insensitive currents. Single-channel recordings were performed in the outside-out configuration using the same solutions, but with glass electrodes of 5-10 M of open resistance. Signals were filtered through a four-pole low-pass Bessel filter at 1 kHz, acquired using pCLAMP 8 and analysed using Bio-Patch analysis software (Bio-Logic Science Instruments, Grenoble, France). The open-state probability (Po) was evaluated at 0 mV, with the assumption that the number of channels present in the patch correspond to the maximum number of superimposed events. Recordings that displayed tolbutamide-resistant single currents were excluded from the analysis. All experiments were performed at room temperature (20-22°C), and the equilibrium potential for K+ ions (EK), calculated after correction of the liquid junction potential, was about -82 mV. Membrane slope conductance values (Gm) were calculated by fitting a straight line to the linear part of the I-V relationship. I-V curves were fitted with Origin 3.5 software (Microcal Software, Inc., Northampton, MA, USA), using a Boltzmann equation of the type: I = Imax/(1 - exp(Vtest - V½/k), where Imax is the maximum current, Vtest the value of the voltage pulse, V½ the voltage at which current is half-activated, and k a slope factor describing the steepness of the curve.
Chemicals
Zinc choride and other inorganic salts (with negligible listed Zn2+ contamination) were obtained from Fluka Chemie AG (Buchs, Switzerland, puriss. p.a. ACS reagent grade). TTX was purchased from Latoxan (Rosans, France). Other drugs and compounds were obtained from Sigma (Sigma-Aldrich Co, Fluka Chemie AG, Buchs, Switzerland). All solutions were prepared in distilled-deionised water to reduce basal Zn2+ levels.
Statistics
Data were expressed as means ± S.E.M. Tests of significance (P < 0·05) were performed using Student's t test (Origin 3.5 software).
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effects of extracellular Zn2+ on RINm5F cell excitability
Under current-clamp conditions, a small percentage of RINm5F cells (< 20 %) with a resting potential of about -40 mV showed spontaneous action potential firing (Fig. 1A). In these cells (n = 4), a discharge of action potentials (maximum frequency, 5 Hz) was recorded in whole-cell configuration during the first minutes after the patch membrane was ruptured. Subsequently, a rapid and progressive run-down in cell excitability occurred. This phenomenon is clearly visible in the trace of Fig. 1A after the period of Ca2+ removal, but it also occurred in cells not submitted to Ca2+ deprivation. Thus, it most probably reflected the wash-out of some intracellular components. The duration of the action potentials (half-width, 20·44 ± 0·30 ms) together with their disappearance in a Ca2+-free medium suggested that they were supported by activation of voltage-dependent Ca2+ channels. Bath perfusion of Zn2+ (10 and 20 µM) also resulted in a total block of action potentials, but in contrast to Ca2+ suppression, this block was accompanied by a hyperpolarisation of the resting membrane potential (Fig. 1A). As shown in Fig. 1B, under voltage-clamp conditions with 1 mM ATP in the patch pipette, application of 10 µM Zn2+ activated a current reversing at a potential below -80 mV. When Zn2+-induced current was plotted against voltage, and fitted using a linear regression (r2 = 0·96), the calculated reversal potential was -82 mV (n = 5), which corresponded to the reversal potential for K+ (EK) in our recording conditions. This effect, which was fully reversible on wash-out of Zn2+, was in addition followed by a transient decrease in the holding current which returned to control level after tens of seconds. This late effect was observed in most of the cells, and also reversed near the equilibrium potential for K+ ions.
 |
View larger version [in this window] [in a new window] |
|
|
A, current-clamp recording from an excitable RINm5F cell, showing spontaneous and repetitive action potential firing. The cell displayed Ca2+ spikes (a portion of the trace is magnified below) which were abolished with the perfusion of a Ca2+-free solution. Bath application of 20 µM Zn2+ totally suppressed Ca2+ spikes and induced a marked hyperpolarization of membrane potential. B, whole-cell currents evoked by bath-applied 10 µM Zn2+, in voltage-clamp mode. Current traces recorded at holding potentials (Vh) from -100 to +20 mV, in 20 mV steps, were superimposed on the onset of Zn2+ applications. The Zn2+-induced current reversed at EK. C, plot of dose-response effect of Zn2+ on membrane slope conductance (Gm), calculated from I-V relationships obtained from voltage ramps from -100 to -60 mV (n = 7-16 cells for each Zn2+ concentration). All the values of Gm in the presence of Zn2+ were significally different (P < 0·01, except for 100 µM Zn2+) compared with control Gm recorded before Zn2+ application. The curve was fitted to the equation | ||
It appeared, therefore, that Zn2+ caused a reversible increase in a voltage-independent K+ membrane conductance, which could be responsible for the hyperpolarisation evoked by Zn2+ that we observed under current-clamp conditions at -40 mV. The Zn2+-induced increase in slope membrane conductance was dose dependent (Fig. 1C), ranging from 19·5 ± 4·8 % (n = 7) for 1 µM Zn2+ to 46·2 ± 9·4 % (n = 16) for 10 µM Zn2+, with a half-maximal effect at 1·7 ± 0·24 µM Zn2+. At higher concentrations this effect showed saturation, since increasing Zn2+ above 10 µM did not result in a further increase in slope membrane conductance (47·1 ± 12·5 % (n = 12), 49·2 ± 14·6 % (n = 10) and 48·8 ± 23·4 % (n = 5), for 20, 50 and 100 µM Zn2+, respectively). Control slope membrane conductance, calculated on a total of 57 RINm5F cells between -100 and -60 mV, was 12·2 ± 1·6 nS.
These results led us to test whether Zn2+ could modify both the voltage-dependent currents involved in the generation of the burst of action potentials and the K+ membrane conductances controlling the resting membrane potential.
Effects of extracellular Zn2+ on voltage-dependent Ca2+ channels
RINm5F cells are known to express multiple high voltage-activated Ca2+ channels, including L-, N- and P/Q types (Magnelli et al. 1995). Under ionic conditions that minimised K+ currents and blocked voltage-dependent Na+ currents, depolarizing steps from a holding potential of -80 mV elicited Ca2+ inward currents (Fig. 2A), which were blocked by application of 250 µM cadmium (not shown). In response to depolarising steps of low amplitude (between about -40 and -10 mV), a rapidly inactivating transient current occurred. Furthermore, upon depolarization with a voltage ramp (Fig. 2B), a deflection in the current trace was evident for voltages between about -45 and -15 mV (-44 ± 0·9 and -16 ± 0·8 mV, n = 15). Both this deflection and the transient Ca2+ current component suggested activation of low voltage-activated Ca2+ channels (LVA). High voltage-activated Ca2+ channels (HVA) were also activated, showing a slower inactivation rate and reaching maximal amplitude at about +10 mV. Zn2+ application (1-500 µM) resulted in a reversible and dose-dependent reduction in the amplitude of both types of Ca2+ currents (Fig. 2C, D and E). However, Zn2+-induced inhibition was more pronounced at -30 mV than at +10 mV. With 10 and 100 µM Zn2+, respectively, Ca2+ currents were blocked by 30·4 ± 3·0 % (n = 14) and 57·0 ± 7·5 % (n = 4) at -30 mV, but only by 12·2 ± 0·9 % (n = 12) and 24·6 ± 1·4 % (n = 4) at +10 mV (Fig. 2C, D and E). Furthermore, 10 µM Zn2+ fully abolished the deflection in current traces arising from LVA currents, although it had little effect on the peak amplitude of HVA currents (Fig. 2B). Thus, LVA channels showed a greater sensitivity than HVA channels to Zn2+ block.
 |
View larger version [in this window] [in a new window] |
|
|
A, superimposed traces of Ca2+ currents induced by depolarization of a RINm5F cell from a holding potential of -80 mV, in 10 mV increments. In addition to the slowly inactivating current, a transient, rapidly inactivating component was recorded in response to low amplitude voltage steps (-30 to -10 mV in this cell). B, during a 350 ms voltage ramp from -80 to +60 mV, the Ca2+ current displayed a shoulder (between -45 and -15 mV) and a peak at about +5 mV, reflecting successive activation of LVA and HVA Ca2+ channels. Bath application of 10 µM Zn2+ decreased the amplitude of the shoulder, but had very little effect on the peak of the current. Traces were corrected by digital subtraction of the leak current measured in the presence of 250 µM Cd2+. C, effects of increasing Zn2+ concentrations (1 min perfusion for each dose) on peak normalised Ca2+ currents at two potentials measured every 30 s. Wash-out periods of 3 min were allowed between Zn2+ applications. The inhibition of Ca2+ current was more pronounced at -30 mV than at 0 mV, reflecting greater sensitivity of LVA Ca2+ current to Zn2+. Note that the maximum effect is reached after 30 s of Zn2+ application, and reverses completely 1-2 min after wash-out. D, current-voltage curves of Ca2+ currents in the presence of increasing concentrations of Zn2+( | ||
Effects of extracellular Zn2+ on K+ conductances
Voltage-gated currents. As seen in Fig. 1A, the spike potentials in RINm5F cells were followed by a rapid repolarisation and an undershoot phase, suggesting activation of voltage-gated K+ conductances. In voltage-clamp mode, two types of voltage-gated K+ currents were recorded. The total current, activated by depolarising pulses after a prepulse at -120 mV, could be divided into two components: a sustained one, activated by depolarising pulses after a prepulse at -20 mV, and a transient one which was obtained by subtracting the sustained component from the total K+ current (Fig. 3A). Applications of increasing concentrations of Zn2+ (20-200 µM) resulted in a slight, but not significant, inhibition of both types of voltage-activated K+ currents (Fig. 3B and C). This effect was also accompanied by a modest rightward shift of I-V curves. For the sustained K+ current, the control curve was best fitted by a Boltzmann equation (see Methods) with V½ of -10·8 mV. In the presence of 20, 100 and 200 µM Zn2+ the corresponding V½ values were -2·6, -2·2 and -6·8 mV, respectively. For the transient K+ outward current, the V½ values were 8·5 mV for control, and 14·7, 7·1 and 16·8 mV for 20, 100, and 200 µM Zn2+, respectively. Thus, Zn2+ induced only a small alteration in voltage-gated K+ currents, with no clear dose dependency over the range of concentrations tested.
 |
View larger version [in this window] [in a new window] |
|
|
A, superimposed traces of voltage-gated K+ currents elicited in a RINm5F cell. The total current activated by depolarising pulses after a prepulse at -120 mV (1) can be divided into a sustained component (2) activated by depolarising pulses after a prepulse at -20 mV and a transient one (1 - 2) which was obtained by digital subtraction of the sustained component from the total K+ current. Stimulation protocols are presented below the current traces. B, current-voltage plots of the sustained (n = 5, left graph) and the transient K+ current (n = 6, right graph) recorded with and without external Zn2+ ( | ||
KATP currents. Extracellular application of Zn2+ to RINm5F cells resulted in a rise of a linear K+ membrane conductance (Fig. 1B) responsible for a shift in resting membrane potential towards EK as evidenced in current-clamp experiments (Fig. 1A). Since KATP channels support the main conductance regulating membrane potential in insulin-secreting cells, we examined whether the effect of Zn2+ resulted from activation of these channels. In the RINm5F cells tested (n = 12), it was possible to record a resting current, which reversed at EK, and which could be blocked by 100 µM tolbutamide and enhanced by 200 µM diazoxide (not shown). Using a voltage ramp as the voltage-clamp stimulation protocol, we observed that the Zn2+-induced increase in slope membrane conductance was abolished in the presence of tolbutamide (100 µM, n = 4, Fig. 4A). On the other hand, neither extracellular application of K+ channel blockers such as TEA (up to 20 mM) plus 4-AP (up to 2 mM) nor Ca2+ omission in the external solution was able to reduce the Zn2+-induced rise in K+ current (n = 5, not shown), suggesting that the voltage-gated and Ca2+-activated K+ channels were not involved. We thus concluded that the Zn2+-induced increase in membrane conductance in RINm5F cells resulted from activation of KATP channels.
 |
View larger version [in this window] [in a new window] |
|
|
A, effects of tolbutamide on Zn2+-induced increase in membrane conductance. Whole-cell currents elicited by voltage ramps from -120 to 80 mV in 5 s (holding potential, -80 mV) and recorded from the same RINm5F cell. Control (dotted) and Zn2+ (continuous) traces are superimposed. a, in control conditions, perfusion with 20 µM Zn2+ increased a current that reversed at about -80 mV. b, in the presence of the KATP channel blocker tolbutamide (100 µM), application of 20 µM Zn2+ failed to increase the whole-cell current. c, slope membrane conductance, calculated between -120 and -40 mV, was expressed as a percentage of the mean conductance recorded before extracellular application of Zn2+ (20 µM). In the absence of tolbutamide ( | ||
Effects of Zn2+ on excised outside-out patch recordings
Figure 5A illustrates individual channel currents fully sensitive to tolbutamide (400 µM), recorded in an outside-out patch of a RINm5F cell with a quasi-physiological cation gradient. With 1 mM ATP in the patch-pipette, the mean amplitude of currents was reversibly increased by application of 20 µM Zn2+ to the external surface of the patch. The current-voltage relationship for current recorded at negative membrane potentials was linear, but showed an inward rectification at membrane potentials higher than +10 mV (Fig. 5C), as previously described in RINm5F patch membrane by Findlay (1987) under similar ionic conditions. The slope conductance fitted between -40 and 0 mV was 17·03 ± 1·27 pS (n = 6) in control conditions, and 17·22 ± 1·29 pS (n = 3) in the presence of 20 µM Zn2+, indicating that the single channel conductance was not affected by the divalent cation. In contrast, open-state probability transiently increased from 0·089 ± 0·015 to 0·188 ± 0·032 in response to application of 20 µM Zn2+ (n = 18, Fig. 5B).
 |
View larger version [in this window] [in a new window] |
|
|
A, representative single KATP channel currents in the outside-out configuration under a quasiphysiological cation gradient. Recording was obtained from a RINm5F cell membrane patch voltage-clamped at 0 mV. Bath application of 20 µM Zn2+ induced simultaneous opening of six individual channels in this example. Openings were completely suppressed by 400 µM tolbutamide. B, open-state probability (Po) measured over a period of 10 s before, during and 30 s after application of 20 µM Zn2+ in the presence of 1 mM ATP ( | ||
Zn2+-induced activation of KATP depends on intracellular ATP
The Zn2+-induced increase in open-state probability of KATP channel was strongly dependent on the presence of ATP in the patch-pipette. Indeed, when ATP was omitted in the pipette solution (Mg-ATP replaced by MgCl2), open-state probability (0·234 ± 0·023, n = 17) became 2·6 times higher than that recorded with an ATP-containing patch pipette, but external application of 20 µM Zn2+ failed to further increase it significantly (0·251 ± 0·027, n = 17, Fig. 5B). The same dependency upon intracellular ATP was observed in whole-cell voltage-clamped RINm5F cells, where the amplitude of the Zn2+-induced increase roughly correlated to the intra-pipette ATP concentration (Fig. 4B). The increase over basal holding current at -40 mV elicited by bath application of 20 µM Zn2+ ranged from 31 ± 1·1 % (n = 11) to 14·3 ± 0·4 % (n = 9) with 1000 and 10 µM ATP, respectively. Total removal of ATP (n = 12) did not further reduce the activating effect of Zn2+. The increase in KATP currents evoked by diazoxide (200 µM), but not their block by tolbutamide (400 µM), was also suppressed upon omission of ATP in the patch pipette (not shown). Thus, when the KATP channel open-state probability was set to its maximal value by reducing intracellular ATP, Zn2+ was no longer efficient in activating the channel.
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Our salient observation here is that Zn2+, at a micromolar concentration, activates KATP currents in rat insulinoma RINm5F cells. In addition, Zn2+, in the tens to hundreds micromolar range, inhibits voltage-gated Ca2+ and, to a lesser extent, K+ currents. Both activation of KATP channels and block of voltage-activated currents were reversible. It is unlikely that the described effects of Zn2+ on voltage-gated channels resulted from screening of bulk membrane-surface negative charges since they were consistently recorded in extracellular medium containing high divalent cation concentrations (12 mM CaCl2 + MgCl2 for Ca2+ currents experiments, and 4 mM CaCl2 + MgCl2 for K+ currents experiments). As KATP channels are not voltage gated, the effects of Zn2+ on KATP currents also cannot be due to changes in membrane surface potential. Rather we suggest that these effects result from binding of Zn2+ to specific amino acid residues of the channel proteins. There is extensive evidence showing that Zn2+ binding alters the behaviour of several ligand-gated ion channels, such as neuronal nicotinic (Palma et al. 1998), ATP-activated (Cloues et al. 1993), glycine-activated (Bloomenthal et al. 1994), GABAA and excitatory amino acid receptors (see Smart et al. 1994). Zn2+ has also been shown to interact with voltage-activated channels, such as voltage-gated Ca2+, K+ or Na+ channels (for review see Harrison & Gibbons, 1994). However, to our knowledge, this is the first report of Zn2+-induced activation of KATP channels.
As previously described in different preparations, we show here that Zn2+ induced a reversible block of voltage-gated Ca2+ channels in RINm5F cells. Pancreatic -cells are known to express both LVA and HVA Ca2+ channels (Ashcroft et al. 1990), and a T-type channel isoform has been recently cloned from INS-1, another rat insulin-secreting cell line (Zhuang et al. 2000). We confirm here that RINm5F cells also express a T-type current in addition to identified HVA channels (Magnelli et al. 1995), and that this current is more sensitive to Zn2+ action than HVA. T-type currents were seen to be more sensitive to Zn2+ than HVA in rat dorsal root ganglia cells (Busselberg et al. 1994), but not in smooth muscle cells (Akaike et al. 1989).
The action of Zn2+ has previously been studied on native and cloned voltage-dependent K+ channels. With the exception of some neuronal, transiently activated currents (Huang et al. 1993; Easaw et al. 1999; and see also Harrison et al. 1993a), these channels are blocked in the presence of micro- to millimolar extracellular Zn2+ in a dose-dependent manner. The divalent cation also shifts both activation and inactivation curves in the depolarising direction, thus slowing the activation kinetics (see Harrison & Gibbons, 1994). We report here that voltage-gated K+ channels expressed by RINm5F cells may also be sensitive to Zn2+, but over the concentration range used (20-200 µM), the inhibition was modest and not clearly dose dependent. This contrasts with the higher sensitivity of the cloned delayed-rectifier and rapidly inactivating human and rat K+ channels, which were potently blocked, and whose activation and inactivation behaviour was clearly altered over the same range of Zn2+ concentrations (Harrison et al. 1993b).
Even though the blocking effects of extracellular Zn2+ on voltage-gated Ca2+ currents (supporting the rising phase of action potentials) and voltage-gated K+ currents (supporting repolarisation) could be responsible for some decrease in RINm5F cells excitability, we do not favour this interpretation. Indeed, abolition of action potential firing occurred with Zn2+ concentrations as low as 10-20 µM, which resulted only in a very modest, if any, block of voltage-activated Ca2+ and K+ channels. Furthermore, alteration of voltage-dependent conductances could not explain the Zn2+-induced hyperpolarization observed in current-clamp mode. We instead propose that extracellular Zn2+ induces a reversible suppression of action potential firing mainly by hyperpolarising the RINm5F cell resting membrane potential following activation of KATP channels. This reversible effect exhibits high specificity (half-maximal activation with 1·7 µM Zn2+), and no voltage dependency. In addition, upon Zn2+ wash-out, we observed a transient decrease in membrane conductance, which apparently reflected an inhibition of KATP currents. Indeed, this late change in conductance reversed near the equilibrium potential for K+ ions, and was sensitive to tolbutamide (not shown). This transient inhibition of KATP current could result from either a 'rebound effect' upon Zn2+ removal, or the occurrence of a slowly reversible block of KATP channels also induced by Zn2+. The latter would be concomitant to its activator effect, but clearly unmasked only upon wash-out of the divalent cation. Such a possible paradoxical action of Zn2+ remains to be investigated.
Single channel analysis reveals that Zn2+-induced activation of KATP current arises from a 2·1-fold increase in KATP channel open-state probability with no change in single channel conductance. Furthermore, the rapid onset and recovery from stimulation of KATP current by Zn2+, together with the fact that the effect is still present in excised patch membranes, suggests that Zn2+ must act directly on KATP channels. Zn2+ binding to one of the subunits forming the channel, most probably at an extracellular site, is an attractive hypothesis.
Our results show also that Zn2+-induced activation of KATP channel opening is dependent on the presence of intracellular Mg-ATP both in whole-cell and excised membrane patch recordings. At first glance, it can be proposed that activation of channel gating is achieved by extracellular Zn2+, only when the open-state probability is first reduced by internal ATP4-. Alternatively, binding of ATP to the channel may be a prerequisite for the Zn2+ binding site to be accessible. Binding of the nucleotide would induce a shift in charged residues, resulting in a conformational change such that the extracellular site for Zn2+ binding become accessible. It is known that the KATP channel is an octameric complex formed by the physical association of two functionally distinct subunits (Inagaki et al. 1995): an inwardly rectifying K+ channel (Kir6.x) and a sulphonylurea binding protein (SUR), with a (Kir6.x-SUR)4 stoichiometry (Clement et al. 1997). Kir6.x subunits are supposed to form the conducting pore for K+ ions and to bear the primary site for the inhibitory action of ATP4-, while SUR subunits, which are members of the ATP-binding cassette protein family, would confer both sensitivity to openers and blockers, and to Mg-nucleotides (see Babenko et al. 1998; Seino, 1999). Thus, according to our last hypothesis, the question remains as to whether Zn2+ action requires previous interaction of Mg-ATP with nucleotide-binding folds located on SUR subunits, or previous binding of ATP4- on the inhibitory site located onto Kir6.x subunits. Further experiments are needed to address this question, in order to delineate possible mechanisms of action of Zn2+ on KATP channels. In contrast, Zn2+ is inefficient in activating KATP channels previously closed by tolbutamide, suggesting either that tolbutamide-induced conformational changes mask the Zn2+-binding site or that the tolbutamide block overcomes the stimulating effect of Zn2+.
It is worth noting that, in contrast to our results, Kwok & Kass (1993) observed a block of cardiac KATP channels by low concentrations of extracellular divalent cations, including Zn2+ (Kd for Zn2+, 0·46 µM). In this study, the degree of cation-induced block was also dependent on intracellular ATP concentrations. Cardiac KATP channels are believed to be composed of Kir6.2 plus SUR2A subunits, whereas in pancreatic -cells (and neurons) Kir6.2 is thought to combine with the SUR1 isoform of sulphonylurea receptors to constitute the channel (see Babenko et al. 1998; Seino, 1999). The same Kir6.2 plus SUR1 combination is expressed in RINm5F cells, attesting to their pancreatic -cell origin (Aguilar-Bryan et al. 1995; Miller et al. 1999). One appealing hypothesis is that Zn2+ could induce opposite effects on KATP currents depending on subunit composition of the channel. This possibility remains to be investigated, as does the functional significance of such a differential control by Zn2+. Comparison of the properties of cloned and wild-type KATP channels indicates that the neuronal channel sub-type is formed by the same subunit combination (Kir6.2 plus SUR1) as the pancreatic -cell channel sub-type (see Babenko et al. 1998; Seino, 1999). Since Zn2+ is concentrated in excitatory nerve terminal synaptic vesicles of many brain regions and released upon nerve activity, it would thus be of interest to examine whether Zn2+ also regulates neuronal excitability through a control over neuronal KATP channels. This could have potential implications for interpretation of the neuromodulatory or neurotoxic actions of Zn2+.
In pancreatic -cells of all but a few species, insulin is believed to be stored inside secretory vesicles as a solid hexamer bound with two Zn2+ ions per hexamer (see Blundell et al. 1971; Dodson & Steiner, 1998). However, measurements of the granular Zn2+:insulin ratio have shown that the Zn2+ concentration is in excess of that required to form (2 Zn2+)-insulin hexamers (Hutton et al. 1983; Foster et al. 1993). This extra Zn2+ has been proposed to stabilise the assembly within the granule (Smith et al. 1984). During exocytosis, secretory vesicles fuse with the plasma membrane, exposing the vesicular interior to the extracellular milieu, and allowing release of stored insulin upon dissolution of Zn2+-insulin crystals. Little is known about the mechanism or time scale of dissolution and dissociation, although Gold & Grodsky (1984) have demonstrated that insulin is free from Zn2+ in less than 60 s after secretion. Release of extragranular Zn2+ has also been observed independently of insulin secretion, and shown to be highly regulated by secretagogues (Formby et al. 1984). Thus, Zn2+ release kinetics are not related solely to insulin secretion, and the free Zn2+ concentration in the extracellular space around -cells at rest and during granule exocytosis is most probably prone to great variations. Since KATP and voltage-activated Ca2+ channels serve as essential regulation of stimulus-secretion coupling in pancreatic -cells, it is reasonable to expect that a Zn2+-induced control of KATP and Ca2+ currents must have some important consequences on -cell functions. In light of our results, the tentative conclusion is that Zn2+ could influence insulin secretion from pancreatic -cells through negative autocrine or paracrine feedback loops, involving both voltage-gated and KATP conductances. This view is consistent with the data reported by Ferrer et al. (1984) and Ghafghazi et al. (1981) showing inhibition of glucose-induced electrical activity and insulin release from murine pancreatic -cells by Zn2+. Our findings also lead us to speculate that feedback control of ionic permeabilities by endogenous Zn2+ secretion might be involved in the generation of the characteristic pattern of -cells responses to glucose stimulation, consisting of cyclic oscillations in membrane potential, on which action potential bursts are superimposed (see Ashcroft & Rorsman, 1989). As to whether impairment of the Zn2+-evoked control of ion channels could be of some clinical interest now remains to be evaluated.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P., Boyd, A. E., Gonzalez, G., Herrera-Sosa, H., Nguy, K., Bryan, J. & Nelson, D. A. (1995). Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268, 423-426 | [Medline] |
| Akaike, N., Kanaide, H., Kuga, T., Nakamura, M., Sadoshima, J. & Tomoike, H. (1989). Low-voltage-activated calcium current in rat aorta smooth muscle cells in primary culture. The Journal of Physiology 416, 141-160 | [Medline] |
| Ashcroft, F. M., Kelly, R. P. & Smith, P. A. (1990). Two types of Ca channel in rat pancreatic beta-cells. Pflügers Archiv 415, 504-506 | |
| Ashcroft, F. M. & Rorsman, P. (1989). Electrophysiology of the pancreatic beta-cell. Progress in Biophysics and Molecular Biology 54, 87-143 | [Medline] |
| Aspinwall, C. A., Brooks, S. A., Kennedy, R. T. & Lakey, J. R. (1997). Effects of intravesicular H+ and extracellular H+ and Zn2+ on insulin secretion in pancreatic beta cells. Journal of Biological Chemistry 272, 31308-31314 | [Abstract/Full Text] |
| Assaf, S. Y. & Chung, S. H. (1984). Release of endogenous Zn2+ from brain tissue during activity. Nature 308, 734-736 | [Medline] |
| Babenko, A. P., Aguilar-Bryan, L. & Bryan, J. (1998). A view of sur/KIR6.X, KATP channels. Annual Review of Physiology 60, 667-687 | [Abstract/Full Text] |
| Bloomenthal, A. B., Goldwater, E., Pritchett, D. B. & Harrison, N. L. (1994). Biphasic modulation of the strychnine-sensitive glycine receptor by Zn2+. Molecular Pharmacology 46, 1156-1159 | [Abstract] |
| Blundell, T., Dodson, G., Hodgkin, D. & Mercola, D. (1971). Insulin: the structure in the crystal and its reflection in chemistry and biology. Advances in Protein Chemistry 26, 279-402. | |
| Busselberg, D., Pekel, M., Michael, D. & Platt, B. (1994). Mercury (Hg2+) and zinc (Zn2+): two divalent cations with different actions on voltage-activated calcium channel currents. Cell and Molecular Neurobiology 14, 675-687. | |
| Choi, D. W. & Koh, J. Y. (1998). Zinc and brain injury. Annual Review of Neuroscience 21, 347-375 | [Abstract/Full Text] |
| Clement, J. P., Kunjilwar, K., Gonzalez, G., Schwanstecher, M., Panten, U., Aguilar-Bryan, L. & Bryan, J. (1997). Association and stoichiometry of K(ATP) channel subunits. Neuron 18, 827-838 | [Medline] |
| Cloues, R., Jones, S. & Brown, D. A. (1993). Zn2+ potentiates ATP-activated currents in rat sympathetic neurons. Pflügers Archiv 424, 152-158 | |
| Dodson, G. & Steiner, D. (1998). The role of assembly in insulin's biosynthesis. Current Opinion in Structural Biology 8, 189-194 | [Medline] |
| Dunant, Y., Loctin, F., Vallee, J. P., Parducz, A., Lesbats, B. & Israel, M. (1996). Activation and desensitisation of acetylcholine release by zinc at Torpedo nerve terminals. Pflügers Archiv 432, 853-858 | |
| Easaw, J. C., Jassar, B. S., Harris, K. H. & Jhamandas, J. H. (1999). Zinc modulation of ionic currents in the horizontal limb of the diagonal band of Broca. Neuroscience 94, 785-795 | [Medline] |
| Ferrer, R., Soria, B., Dawson, C. M., Atwater, I. & Rojas, E. (1984). Effects of Zn2+ on glucose-induced electrical activity and insulin release from mouse pancreatic islets. American Journal of Physiology 246, C520-527 | [Medline] |
| Findlay, I. (1987). The effects of magnesium upon adenosine triphosphate-sensitive potassium channels in a rat insulin-secreting cell line. The Journal of Physiology 391, 611-629 | [Medline] |
| Formby, B., Schmid-Formby, F. & Grodsky, G. M. (1984). Relationship between insulin release and 65zinc efflux from rat pancreatic islets maintained in tissue culture. Diabetes 33, 229-234 | [Medline] |
| Foster, M. C., Leapman, R. D., Li, M. X. & Atwater, I. (1993). Elemental composition of secretory granules in pancreatic islets of Langerhans. Biophysical Journal 64, 525-532 | [Abstract] |
| Ghafghazi, T., Mcdaniel, M. L. & Lacy, P. E. (1981). Zinc-induced inhibition of insulin secretion from isolated rat islets of Langerhans. Diabetes 30, 341-345 | [Medline] |
| Gold, G. & Grodsky, G. M. (1984). Kinetic aspects of compartmental storage and secretion of insulin and zinc. Experientia 40, 1105-1114 | [Medline] |
| Harrison, N. L. & Gibbons, S. J. (1994). Zn2+: an endogenous modulator of ligand- and voltage-gated ion channels. Neuropharmacology 33, 935-952 | [Medline] |
| Harrison, N. L., Radke, H. K., Talukder, G. & Ffrench-Mullen, J. M. (1993a). Zinc modulates transient outward current gating in hippocampal neurons. Receptors and Channels 1, 153-163 | [Medline] |
| Harrison, N. L., Radke, H. K., Tamkun, M. M. & Lovinger, D. M. (1993b). Modulation of gating of cloned rat and human K+ channels by micromolar Zn2+. Molecular Pharmacology 43, 482-486 | [Abstract] |
| Howell, G. A., Welch, M. G. & Frederickson, C. J. (1984). Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308, 736-738 | [Medline] |
| Huang, R. C., Peng, Y. W. & Yau, K. W. (1993). Zinc modulation of a transient potassium current and histochemical localization of the metal in neurons of the suprachiasmatic nucleus. Proceedings of the National Academy of Sciences of the USA 90, 11806-11810 | [Abstract] |
| Hutton, J. C., Penn, E. J. & Peshavaria, M. (1983). Low-molecular-weight constituents of isolated insulin-secretory granules. Bivalent cations, adenine nucleotides and inorganic phosphate. Biochemical Journal 210, 297-305 | [Medline] |
| Inagaki, N., Gonoi, T., Clement, J. P., Namba, N., Inazawa, J., Gonzalez, G., Aguilar-Bryan, L., Seino, S. & Bryan, J. (1995). Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270, 1166-1170 | [Abstract] |
| Kwok, W. M. & Kass, R. S. (1993). Block of cardiac ATP-sensitive K+ channels by external divalent cations is modulated by intracellular ATP. Evidence for allosteric regulation of the channel protein. Journal of General Physiology 102, 693-712 | [Abstract] |
| Magnelli, V., Pollo, A., Sher, E. & Carbone, E. (1995). Block of non-L-, non-N-type Ca2+ channels in rat insulinoma RINm5F cells by omega-agatoxin IVA and omega-conotoxin MVIIC. Pflügers Archiv 429, 762-771 | |
| Miller, T. R., Taber, R. D., Molinari, E. J., Whiteaker, K. L., Monteggia, L. M., Scott, V. E., Brioni, J. D., Sullivan, J. P. & Gopalakrishnan, M. (1999). Pharmacological and molecular characterization of ATP-sensitive K+ channels in the TE671 human medulloblastoma cell line. European Journal of Pharmacology 370, 179-185 | [Medline] |
| Nishimura, M. (1988). Zn2+ stimulates spontaneous transmitter release at mouse neuromuscular junctions. British Journal of Pharmacology 93, 430-436 | [Medline] |
| Palma, E., Maggi, L., Miledi, R. & Eusebi, F. (1998). Effects of Zn2+ on wild and mutant neuronal alpha7 nicotinic receptors. Proceedings of the National Academy of Sciences of the USA 95, 10246-10250 | [Abstract/Full Text] |
| Perez-Clausell, J. & Danscher, G. (1985). Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study. Brain Research 337, 91-98 | [Medline] |
| Praz, G. A., Halban, P. A., Wollheim, C. B., Blondel, B., Strauss, A. J. & Renold, A. E. (1983). Regulation of immunoreactive-insulin release from a rat cell line (RINm5F). Biochemical Journal 210, 345-352 | [Medline] |
| Seino, S. (1999). ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annual Review of Physiology 61, 337-362 | [Abstract/Full Text] |
| Slomianka, L. (1992). Neurons of origin of zinc-containing pathways and the distribution of zinc-containing boutons in the hippocampal region of the rat. Neuroscience 48, 325-352 | [Medline] |
| Smart, T. G., Xie, X. & Krishek, B. J. (1994). Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc. Progress in Neurobiology 42, 393-341 | [Medline] |
| Smith, G. D., Swenson, D. C., Dodson, E. J., Dodson, G. G. & Reynolds, C. D. (1984). Structural stability in the 4-zinc human insulin hexamer. Proceedings of the National Academy of Sciences of the USA 81, 7093-7097 | [Medline] |
| Vallee, B. L. & Falchuk, K. H. (1993). The biochemical basis of zinc physiology. Physiological Reviews 73, 79-118 | [Medline] |
| Vautrin, J., Schaffner, A. E. & Barker, J. L. (1993). Tonic GABA secretion of cultured rat hippocampal neurons rapidly transformed by Zn2+ into quantal release. Neuroscience Letters 158, 125-129 | [Medline] |
| Wang, Y. X. & Quastel, D. M. (1990). Multiple actions of zinc on transmitter release at mouse end-plates. Pflügers Archiv 415, 582-587 | |
| Xie, X. M. & Smart, T. G. (1991). A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission. Nature 349, 521-524 | [Medline] |
| Zhuang, H., Bhattacharjee, A., Hu, F., Zhang, M., Goswami, T., Wang, L., Wu, S., Berggren, P. O. & Li, M. (2000). Cloning of a T-type Ca2+ channel isoform in insulin-secreting cells. Diabetes 49, 59-64 | [Medline] |
This work was supported by a grant from the Swiss NFSR (no. 31-057135.99) to A.B. and Y.D., T.C. was a recipient of postdoctoral long-term fellowship from the EMBO (no. ALTF 529-1997). We would like to thank C. B. Wollheim and D. Muller for helpful discussions, F. Loctin for excellent technical assistance, L. S. Jones for detailed revision of the manuscript, and W. Pralong and A. Robert for initiating this research.
A. Bloc and T. Cens contributed equally to this paper.
Corresponding author
A. Bloc: APSIC-Pharmacologie, Centre Médical Universitaire, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland.
Email: alain.bloc{at}medecine.unige.ch
Author's present address
T. Cens: CRBM, CNRS UPR 1086, 1919 Route de Mende, 34293 Montpellier, France.
This article has been cited by other articles:
|
|
 |
|
  Y. Zhao, Q. Fang, S. G. Straub, and G. W. G. Sharp Both Gi and Go Heterotrimeric G Proteins Are Required to Exert the Full Effect of Norepinephrine on the {beta}-Cell KATP Channel J. Biol. Chem., February 29, 2008; 283(9): 5306 - 5316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhou, T. Zhang, J. S. Harmon, J. Bryan, and R. P. Robertson Zinc, Not Insulin, Regulates the Rat {alpha}-Cell Response to Hypoglycemia In Vivo Diabetes, April 1, 2007; 56(4): 1107 - 1112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gromada, I. Franklin, and C. B. Wollheim {alpha}-Cells of the Endocrine Pancreas: 35 Years of Research but the Enigma Remains Endocr. Rev., February 1, 2007; 28(1): 84 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Chimienti, S. Devergnas, F. Pattou, F. Schuit, R. Garcia-Cuenca, B. Vandewalle, J. Kerr-Conte, L. Van Lommel, D. Grunwald, A. Favier, et al. In vivo expression and functional characterization of the zinc transporter ZnT8 in glucose-induced insulin secretion J. Cell Sci., October 15, 2006; 119(20): 4199 - 4206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Takahashi, H. Ishihara, A. Tamura, S. Yamaguchi, T. Yamada, D. Takei, H. Katagiri, H. Endou, and Y. Oka Cell type-specific activation of metabolism reveals that {beta}-cell secretion suppresses glucagon release from {alpha}-cells in rat pancreatic islets Am J Physiol Endocrinol Metab, February 1, 2006; 290(2): E308 - E316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Munoz, M. Hu, K. Hussain, J. Bryan, L. Aguilar-Bryan, and A. S. Rajan Regulation of Glucagon Secretion at Low Glucose Concentrations: Evidence for Adenosine Triphosphate-Sensitive Potassium Channel Involvement Endocrinology, December 1, 2005; 146(12): 5514 - 5521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Marthinet, A. Bloc, Y. Oka, Y. Tanizawa, B. Wehrle-Haller, V. Bancila, J.-M. Dubuis, J. Philippe, and V. M. Schwitzgebel Severe Congenital Hyperinsulinism Caused by a Mutation in the Kir6.2 Subunit of the Adenosine Triphosphate-Sensitive Potassium Channel Impairing Trafficking and Function J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5401 - 5406. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Nikonenko, M. Bancila, A. Bloc, D. Muller, and P. Bijlenga Inhibition of T-Type Calcium Channels Protects Neurons from Delayed Ischemia-Induced Damage Mol. Pharmacol., July 1, 2005; 68(1): 84 - 89. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Franklin, J. Gromada, A. Gjinovci, S. Theander, and C. B. Wollheim {beta}-Cell Secretory Products Activate {alpha}-Cell ATP-Dependent Potassium Channels to Inhibit Glucagon Release Diabetes, June 1, 2005; 54(6): 1808 - 1815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Bancila, T. Cens, D. Monnier, F. Chanson, C. Faure, Y. Dunant, and A. Bloc Two SUR1-specific Histidine Residues Mandatory for Zinc-induced Activation of the Rat KATP Channel J. Biol. Chem., March 11, 2005; 280(10): 8793 - 8799. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-L. Prost, A. Bloc, N. Hussy, R. Derand, and M. Vivaudou Zinc is both an intracellular and extracellular regulator of KATP channel function J. Physiol., August 15, 2004; 559(1): 157 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yokoi, N. G. Egger, V. M. S. Ramanujam, N. W. Alcock, H. H. Dayal, J. G. Penland, and H. H. Sandstead Association between plasma zinc concentration and zinc kinetic parameters in premenopausal women Am J Physiol Endocrinol Metab, November 1, 2003; 285(5): E1010 - E1020. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Gm = GmaxS/(S½ + S), where 
, no external Zn2+; 
, 5 µM Zn2+; 
, 50 µM Zn2+; 
, 500 µM Zn2+). E, dose-response effect of increasing concentrations of Zn2+ on Ca2+ current at two potentials. The block of Ca2+ current at +10 mV (
) needed higher concentrations of Zn2+ than the one at -30 mV (
, 100 µM Zn2+; 
, 200 µM Zn2+). Curves were fitted as described in Methods. C, histograms showing the normalised amplitude of the sustained (left panel) and the transient K+ current (right panel) recorded at +40 mV, with and without external Zn2+ (
, no external Zn2+; 
, 20 µM Zn2+; 
, 100 µM Zn2+; 
, 200 µM Zn2+). In both cases the changes were not significant. Symbols or columns and error bars indicate means and S.E.M.
, dashed curve) or absence (